O. Kensch et al., HIV-1 reverse transcriptase-pseudoknot RNA aptamer interaction has a binding affinity in the low picomolar range coupled with high specificity, J BIOL CHEM, 275(24), 2000, pp. 18271-18278
Systematic evolution of ligands by exponential enrichment (SELEX) is a powe
rful method for the identification of small oligonucleotides that bind with
high affinity and specificity to target proteins. Such DNAs/RNAs are a new
class of potential chemotherapeutics that could block the enzymatic activi
ty of pathologically relevant proteins. We have conducted a detailed bioche
mical study of the interaction of human immunodeficiency virus I (HIV-1) re
verse transcriptase (RT) with a SELEX-derived pseudoknot RNA aptamer. Elect
ron paramagnetic resonance spectroscopy of site-directed spin-labeled RT mu
tants revealed that this aptamer was selected for binding to the "closed" c
onformation of the enzyme. Kinetic analysis showed that the RNA inhibitor b
ound to HIV RT in a two-step process, with association rates similar to tho
se described for model DNA/DNA and DNA/RNA substrates. However, the dissoci
ation of the pseudoknot RNA from RT was dramatically slower than observed f
or model substrates. Equilibrium binding studies revealed an extraordinaril
y low K-d, of about 25 PM, for the enzyme-aptamer interaction, presumably a
consequence of the slow off-rates. Additionally, this pseudoknot aptamer i
s highly specific for HIV-1 RT, with the closely related HIV-2 enzyme showi
ng a binding affinity close to 4 orders of magnitude lower.